The following discussion of the background to the invention is included to explain the context of the invention. This is not to be taken as an admission that any of the material referred to was published, known or part of the common general knowledge in Australia as at the priority date of any of the claims.
Analytical samples can be characterised and analysed by spectrophotometric measurements of the absorption of light by a sample. Analytical samples can also be characterised and analysed by spectrophotometric measurements of the fluorescence of a sample.
To perform a spectrophotometric measurement of the absorption of light by a sample a source of substantially monochromatic light of a selected wavelength is provided for the purpose of illuminating an analytical sample. Such substantially monochromatic light is conveniently obtained by providing a continuum light source such as a xenon arc lamp or flash lamp and also providing a wavelength selective means such as a grating monochromator between the continuum light source and the analytical sample. A means of detecting and measuring the intensity of the substantially monochromatic light after its passage through an analytical sample in an appropriate sample container is also provided so that the absorption of light by the analytical sample can be measured.
To perform a measurement of the fluorescence of a sample a source of substantially monochromatic light of a first wavelength is similarly provided for illuminating an analytical sample and causing the sample to emit light. The substantially monochromatic light of a first wavelength that illuminates the analytical sample is called the excitation light, and the light emitted by the illuminated analytical sample is called the emission light. A means of selecting from the emission light substantially monochromatic light of a second wavelength is provided and this light is transmitted to a light detecting device for detection and measurement. Such selecting means can be for example a second grating monochromator between the analytical sample and the light detecting device.
Light detecting devices useful in spectrophotometers include photomultiplier tubes, photodiodes and charge-coupled devices. All such devices produce an electrical signal that is proportional to the quantity of light (i.e. to the number of photons per second) reaching the device. It is a characteristic of such light detecting devices that the signal-to-noise ratio is less when the quantity of light failing on the device is less, provided that the quantity of detected light is always sufficiently low that the detecting device is able to operate correctly. In practice, the design of the spectrophotometer ensures that the quantity of detected light is always kept sufficiently low that the detecting device is able to operate correctly. Consequently, the best signal-to-noise ratio is achieved when as much detectable light as possible reaches the detecting device.
To achieve the best signal-to-noise ratio the analytical sample should be uniformly illuminated with the required substantially monochromatic light, and such uniform illumination ideally should be achieved while allowing all the available substantially monochromatic light to enter the sample container and interact with the sample. Any of said light that does not enter the sample container is wasted, and the signal-to-noise ratio of the measurement is less than it might otherwise be. Similarly, it is also desirable that light of interest transmitted through or emitted by the illuminated sample be efficiently collected and transmitted to the light detecting device. Any light of interest that is not collected and transmitted to the light detecting device is wasted, and the signal-to-noise ratio of the measurement is less than it might otherwise be.
Liquid analytical samples are advantageously presented to a spectrophotometer with the aid of a device called a “microplate” or “well plate”. These terms are synonymous; for convenience, the term “well plate” will be used herein. A well plate consists of a multiplicity of sample containers rigidly mounted in an array. Movement of the array with respect to the optical path in the spectrophotometer allows each sample in turn to be illuminated with appropriate substantially monochromatic light so that light of interest can be detected and measured. In the case of absorption measurements the light of interest will be light that has passed through the sample. In the case of fluorescence measurements the light of interest will be light that has been emitted by the illuminated sample. This arrangement allows rapid and convenient analysis of a large number of individual analytical samples. A spectrophotometer arranged to operate in this fashion is known as a well plate reader or microplate reader. In order to provide as many sample containers as possible in a well plate of constant area, it is common to make such sample containers much deeper than they are wide. This long, narrow configuration of the sample container introduces difficulties in the illumination of the sample contained therein. It also introduces difficulties in the collection of light of interest from the sample for detection and measurement.
For example, in prior art well plate readers it is common to illuminate the sample with a cone of substantially monochromatic light formed between the focusing component and the focus. The focus is positioned in the well below the surface of the sample. The subtended angle of the cone of light is made as large as possible to maximise the quantity of light provided. The limitation of this arrangement becomes evident when only a limited volume of sample is available and consequently the surface of the sample is considerably below the top of the well. When the focus is positioned below the surface of the sample the top edges of the well obscure some of the light that is intended for illumination of the sample.
When absorbance measurements are being made any obstruction of light by the top edges of the well inevitably reduces the amount of light reaching the absorbance detector and thereby reduces the signal-to-noise ratio of the absorbance measurements.
When fluorescence measurements are being made the quantity of fluorescently emitted light is proportional to the quantity of light illuminating the sample, so any reduction in the quantity of illuminating light is inevitably associated with a reduction in the quantity of light fluorescently emitted by the sample. This in turn reduces the signal-to-noise ratio of the fluorescence measurements.
The prior-art arrangement for collecting fluorescently emitted light is the same as that just described for illumination. This results in a further shortcoming for fluorescence measurements of a sample having a surface considerably below the top of the well. The optical path defined by the collection angle of the fluorescently-emitted light is obscured by the top of the well. The effective collection angle is thereby reduced. This reduces the quantity of light reaching the fluorescence detector by a factor similar to that by which the illumination of the sample is reduced. This results in a further reduction of the signal-to-noise ratio of the fluorescence measurements.